Microspheres have been used as delivery vehicles for drugs and cells, as they provide a minimally invasive means of transplantation. In particular, many materials and fabrication methods in the delivery of cells for regenerative medicine purposes have been explored because of their advantages: simplicity of large-scale culture of cells in microspheres of controlled sizes, provision of a tunable three-dimensional (3-D) environment for cells, ability to incorporate biochemical signals and biomechanical moieties, as well as simplicity of direct injection of cell-loaded microspheres into defect sites without trypsinization.
Studies were usually done through a two-step method of first fabricating microspheres, for example, through single or double emulsion methods, electrospraying and thermally induced phase separation, and subsequently seeding cells onto them. Although the above-mentioned methods were able to support cells, the microsphere fabrication techniques usually required specialized equipment or a significant amount of time, as thorough washing steps were necessary after chemical-based treatment.
Furthermore, these techniques largely catered for anchorage dependent cells such as fibroblasts. Several other groups reported techniques of direct cell encapsulation into microspheres using either synthetic polyethylene glycol diacrylate, which requires surface modification and addition of enzyme-degradation sites, or natural biopolymers such as alginate, which possess batch-to-batch variation as well as uncontrollable degradation rates.
Tissue engineering techniques generally require the use of a temporary scaffold as a three-dimensional template for initial cell attachment and subsequent tissue formation. The ability of the scaffold to be metabolised by the body allows it to be gradually replaced by new cells to form functional tissue. As such, scaffold design is one of the most important aspects of tissue engineering.
Hydrogels have shown great promise as a scaffold for tissue engineering due to their tissue-like water content, good biocompatibility, and injectable accessibility for in situ grafting. However, substantial challenges remain in the use of hydrogels as scaffold and cell delivery materials. For example, hydrogels have low cell affinity. Therefore, when they are used to encapsulate cells commonly used in regenerative medicine, such as fibroblasts, osteoblasts, endothelial, epithelial and smooth muscle cells, these anchorage dependent cells (ADC) do not spread out in the hydrogel framework but are constrained into a spheroidal shape, thereby leading to poor settlement and frequent occurrence of cell death. In addition, spatial confinement of cells within hydrogel bulk prevents cell migration and cell-cell interaction which are essential in mediating cell differentiation and tissue regeneration, as well as inhibiting cell aggregation which is particularly necessary for the reorganization of tissues, such as cartilage and liver.
The liver is the largest internal organ in human body, responsible for a number of essential functions such as detoxification and protein synthesis. Alcoholism and diseases such as hepatitis account for most acute or chronic liver failures. Currently, tens of millions of people worldwide are suffering from this ailment, but only a small percentage of them receive liver transplants because of a severe shortage of liver donors. Additionally, patients receiving successful liver transplantation do not always have a full recovery. They risk immune-rejections and have life-long dependence on immunosuppressive drugs. The rising prevalence of liver diseases has prompted researchers to search for alternative treatments, such as liver cell transplantation, as possible solutions; these have been extensively explored in the past decade.
Liver cell transplantation relies on the introduction of mature hepatocytes or liver stem cells into the host to restore, maintain or improve defective liver functions. Mature hepatocytes have suboptimal proliferation capacity in vitro and they rapidly lose their phenotype in two-dimensional monolayer cultures. Although hepatocyte transplantation may have an immediate therapeutic effect, its clinical application is limited by the availability and quality of the cells. Studies have reported the maintenance of liver-specific functionalities in three-dimensional culture, when hepatocellular aggregates or spheroids were formed. In this sense, generating liver cell spheroids with controllable size and shape poses one of the key challenges in liver tissue engineering research and development.
Various methodologies have been explored to aid the formation of these spheroids. Common approaches include using bioreactors, photolithography or micropatterning to create molds of appropriate sizes. Nonetheless, these approaches require specialized equipment in order to generate spheroids of controllable size and have faced considerable difficulties in scaling-up.
In view of the above, there remains a need for methods of forming hydrogel microparticles, which may be used in compositions for the manufacture of scaffolds for tissue engineering, which addresses one or more of the above-mentioned issues.